The effects of hypoxia on tumor physiology and response to radiation therapy have been extensively described using in vitro studies, pre-clinical models and in human tumors. Investigations regarding the modulation of molecular signaling by hypoxia are less advanced, with the conclusions of the majority of published studies performed in cellular or subcellular systems. Despite the observation of abnormal blood vessels and regions of pseudopalisading necrosis in high-grade glial brain tumors, little attention has been given to the role of hypoxia in the mechanism of their progression. Recently however, the central role of hypoxia in brain tumor progression and aggressiveness has been hypothesized based on pathological examination of WHO Grade 4 glial tumors by Brat et al. Their studies and conclusions were based upon HIF-1? staining in regions of pseudopalisading necrosis. We have developed the methodology to directly and more specifically measure hypoxia in tissue from all grades of human brain cancer using biopsy-based immunohistochemical (IHC) analysis of EF5 binding. The current application proposes to extend our biopsy-based analyses to PET studies of [F-18]-EF5 and compare patient outcome with [F-18]-EF5 data and IHC-based endpoints (Specific Aim 1). In our second Specific Aim, the tissues obtained from these patients, as well as tissues archived from patients previously treated with EF5 will be used to study the physiologic and molecular hypoxia-dependent mechanisms that result in tumor resistance or cellular hypoxia tolerance. Analyses using software for fluorescence images, developed in our lab, will be performed on frozen sections stained for EF5, PECAM/CD31 (blood vessels), TUNEL (apoptosis) and Ki67 (proliferation). In our third Specific Aim, tissue microarrays (TMAs) will be constructed to assess the impact of microenvironment on molecular signaling pathways. At the completion of this grant we will have compared a validated, quantitative IHC-based marker of hypoxia (EF5) to the non-invasive assay using the identical compound ([18-F]-EF5 PET) and determined the mechanisms and situations under which hypoxia is important in human glial tumors, based upon clinical outcome. [unreadable] [unreadable]